The goal of this proposal is to further understand the interaction of radiation with the DNA molecule. The replicative intermediate of bacteriophage X174 DNA synthesis will be used. This is a well defined double-stranded, covalently closed, circular DNA (CCC-DNA) of 3.4 million molecular weight. It has nine genes, all of which are mapped and their functions identified. Single-strand breaks are easily detected as the conversion of the CCC-DNA to open circular DNA on neutral sucrose gradients. Biological activity of the DNA is measured by the production of mature phage in spheroplasts or calcium-treated cells. The mature phage DNA is single stranded and circular. It also can be easily assayed for radiation damage resulting in strand breaks and loss of biological activity. The role played by the primary water radicals (electron, hydrogen atom and hydroxyl radical) will be studied by irradiation in dilute aqueous solution in the presence of radical scavengers. Conditions which may favor repair of the radiobiological damage will be studied. Possible radiation-induced alterations in the fragmentation pattern of the DNA by restriction endonucleases will be investigated. We also intend to determine if any of the nine genes is particularly sensitive to radiation damage. The long-term goal is to understand the mechanisms of radiobiological action on DNA with the hope that this will help in the intelligent design of experiments with whole animals.